Protein translation by the ribosome is essential for cellular life but the molecular mechanisms governing how ribosomes are made are poorly defined. Ribosome biogenesis is a major consumer of cellular energy and an incredibly complex pathway involving hundreds of trans-acting factors. Ribosome assembly is regulated by well-known tumor suppressors like p53 as well as protoncogenes such as cMyc and TOR, and is emerging in the field as a new target for cancer therapy. Currently we are taking a structure/function approach to decipher the roles of several protein complexes including Nsa1/Rix7 and Grc3/Las1 that are essential for ribosome assembly and cell viability. Nsa1 (WDR74 in mammals) and Rix7 (NVL2 in mammals) are two assembly factors required for production of the large ribosomal subunit. Rix7 is a type-II double ring AAA-ATPase that shares significant homology with the well-studied CDC48/p97. Moreover Rix7s mammalian homologue NVL2 had been linked to cancer and mental illness disorders, highlighting the need understand how the ATPase works. Nsa1 binds to late-stage nucleolar pre-60S ribosome particles and its release from pre-60S particles is thought to be driven by Rix7. We utilized yeast genetics approaches to demonstrate that both the D1 and D2 AAA domains of Rix7 are important for cell viability and ribosome production. We utilized cryo-EM to determine the three dimensional architecture of the D1 and D2 domains to near atomic resolution. The tandem AAA domains form an asymmetric stacked homohexameric ring. Through use of an ATP hydrolysis deficient mutant, we trapped Rix7 with a polypeptide engaged in the central channel formed by the D1 and D2 rings. The presence of this peptide revealed Rix7s role as a molecular unfoldase. Las1 was recently discovered as the long-sought after endoribonuclease which cleaves at the C2 site within the internal transcribed spacer 2 (ITS2) during ribosome assembly. Cleavage at the C2 site is an essential step during ribosome assembly because it separates the precursors of the 5.8S and 25S rRNA, triggers the further processing of the 5 end of the 26S pre-rRNA, and primes pre-60S particles for transit from the nucleolus to the nucleoplasm. While it is now established that Las1 is the C2 endoribonuclease the molecular mechanisms governing C2 cleavage are unclear. Mutations in the mammalian Las1 gene have also been linked to neurological dysfunction, underscoring the need to further understand the activity of this enzyme. Previously we determined that in yeast Las1 is dependent upon its binding partner, the polynucleotide kinase Grc3 (Nol9 in humans) to stimulate specific endonuclease activity. Together Grc3 and Las1 assemble into a tetrameric complex composed of a dimer of Grc3/Las1 heterodimers. We also established that there is functional cross-talk between the nuclease and kinase domains of Las1 and Grc3 allowing for an exquisite level of control of enzymatic activity. After establishing the basic mechanisms for cleavage and phosphorylation in yeast we characterized the human Las1L-Nol9 complex. Through co-immunoprecipitation we established that Las1L and Nol9 assemble into a higher order complex and we identified key regions within each protein that mediate complex assembly. We also used high-resolution imaging to define the spatial pattern of Las1L-Nol9 with the nucleolar sub-structure linked with late pre-rRNA processing events. Finally we discovered that Nol9 encodes of a nucleolar localization sequence that is required for the nucleolar transport of both Las1 and Nol9. Taken together our work establishes shared and distinct features of the Grc3/Las1 complex across eukaryotes.